WO2002060426A2 - Composes regulant la proliferation et la differentiation cellulaires - Google Patents

Composes regulant la proliferation et la differentiation cellulaires Download PDF

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WO2002060426A2
WO2002060426A2 PCT/US2002/000307 US0200307W WO02060426A2 WO 2002060426 A2 WO2002060426 A2 WO 2002060426A2 US 0200307 W US0200307 W US 0200307W WO 02060426 A2 WO02060426 A2 WO 02060426A2
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cells
cell
compounds
neu
proliferation
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PCT/US2002/000307
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WO2002060426A3 (fr
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Philip Leder
Valeria R. Fantin
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President And Fellows Of Harvard College
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Priority to AU2002249913A priority Critical patent/AU2002249913A1/en
Priority to EP02718795A priority patent/EP1349614A2/fr
Publication of WO2002060426A2 publication Critical patent/WO2002060426A2/fr
Publication of WO2002060426A3 publication Critical patent/WO2002060426A3/fr
Priority to US10/613,762 priority patent/US20040097408A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4422—1,4-Dihydropyridines, e.g. nifedipine, nicardipine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Oncogenes are defined as genetically altered genes whose mutated expression product or level of expression somehow disrupts normal cellular function or control (Spandidos et al. (1989) Anticancer Res. 9:821). These types of mutations are believed to have effects on the malignant growth of cells derived from practically every tissue. Oncogenes also include tumor suppressor genes, such as p53 and p53-like genes, whose lack of expression results in uncontrolled cell proliferation.
  • the Neu/HER-2/erbB2 proto-oncogene is a member of the epidermal growth factor (EGF) receptor family.
  • GEF epidermal growth factor
  • Neu is a rodent gene and Her-2 is its human homologue.
  • This group of receptor tyrosine kinases plays an essential role during growth and differentiation of many tissues.
  • overexpression of these proteins is associated with several types of human cancers (reviewed in Hynes et al. (1994) Biochim Biophys Acta 1198(2-3), 165-84; Kim et al. (1999) Exp Cell Res 253(1), 78-87; and Hung et al. (1999) Seminars in Oncology 26 (4 Suppl 12), 51-9).
  • Fig. 12 shows Western blots of lysates from EpH4-A6 and EpH4-EV cells treated for 0, 18 or 36 hours with 3 ⁇ M F16, incubated with anti-pNeu; anti- ⁇ actin; andi-pPKB; anti-PKB; anti-pMAPK and anti-ERK-1 antibodies.
  • the invention is based at least in part on the identification of several small molecules, which inhibit proliferation of transformed cells, without substantially affecting the proliferation of non-transformed cells.
  • the following compounds inhibit proliferation of, or kill, cells over-expressing Neu and/or Ras and/or Myc at very low concentrations, at about 1 ⁇ M or even 100 nM and 10 nM: E20 (ID 141525); F12 (ID 120590); F16 (ID 274873); H10 (ID 120670); J6 (ID 120856); N12 (ID 215015); L4 (ID 121113); Bl 5 (ID 217496) and QR.
  • this invention provides methods for the suppression of oncogenesis that is mediated by the HER-2/c-erb B-2/neu oncogene, an oncogene which has been correlated with a poor prognosis of breast and ovarian carcinoma in humans.
  • the invention also relates to the suppression of oncogenesis mediated by Ras.
  • disorders associated with excessive cellular proliferation resulting from, e.g., infection by a pathogen, such as a virus (e.g., Human Papilloma Virus (HPV)) can also be treated according to the methods of the invention.
  • the compounds of the invention are also useful as markers of certain types of cells, such as cells proliferating excessively, in particular transformed cells. It has been shown herein that cells that are killed by F16 or QR fluoresce before being killed. Thus, F16 and QR can be used not only to kill cells, but also to label cells that will be killed or that are being killed by it.
  • Normalizing cell proliferation refers to reducing the rate of cell proliferation of a cell that proliferates excessively relative to that of its normal or wild-type counterpart, or increasing the rate of cell proliferation of a cell that proliferates poorly relative to its normal or wild-type counterpart.
  • oncogene refers to a gene which is associated with certain forms of cancer. Oncogenes can be of viral origin or of cellular origin. An oncogene is a gene encoding a mutated form of a normal protein or is a normal gene which is expressed at an abnormal level, e.g., over-expressed. Over-expression can be caused by a mutation in a transcriptional regulatory element, e.g., the promoter, or by chromosomal rearrangement resulting in subjecting the gene to an unrelated transcriptional regulatory element.
  • a transcriptional regulatory element e.g., the promoter
  • chromosomal rearrangement resulting in subjecting the gene to an unrelated transcriptional regulatory element.
  • papillomas which refers to benign tumors derived from epithelium and having a papillomavirus as a causative agent; and "epidermoidomas”, which refers to a cerebral or meningeal tumor formed by inclusion of ectodermal elements at the time of closure of the neural groove.
  • keratosis refers to proliferative skin disorder characterized by hyperplasia of the homy layer of the epidermis.
  • exemplary keratotic disorders include keratosis foUicularis, keratosis palmaris et plantaris, keratosis pharyngea, keratosis pilaris, and actinic keratosis.
  • alkyl refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), and more preferably 20 or fewer.
  • preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
  • alkyl (or “lower alkyl) as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • aralkyl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
  • the heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
  • substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,
  • amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula: R 10
  • R9, RI Q and R' J O eacn independently represent a hydrogen, an alkyl, an alkenyl,
  • acylamino is art-recognized and refers to a moiety that can be represented by the general formula:
  • R9, R10 are as defined above.
  • Preferred embodiments of the amide will not include imides which may be unstable.
  • X is a bond or represents an oxygen or a sulfur
  • R ⁇ ⁇ represents a hydrogen, an alkyl, an alkenyl, -(CH2) m -Rg or a pharmaceutically acceptable salt
  • R' ⁇ ⁇ represents a hydrogen, an alkyl, an alkenyl or -(CH2) m -R , where m and Rg are as defined above.
  • alkoxyl or "alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto.
  • Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.
  • An "ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O- alkenyl, -O-alkynyl, -O-(CH2) m -Rg, where m and Rg are described above.
  • triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively.
  • triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.
  • Q represented S or O
  • R46 represents hydrogen, a lower alkyl or an aryl.
  • the phosphoryl group of the phosphorylalkyl can be represented by the general formula:
  • each expression e.g. alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
  • the term "substituted" is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described herein above.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.
  • protecting group means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations.
  • protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively.
  • the field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2 nd ed.; Wiley: New York, 1991).
  • the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes.
  • the prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug.
  • X represents N or CR
  • R represents independently for each occurrence H, halogen, alkyl, alkenyl, alkynyl, hydroxyl, alkoxyl, silyloxy, amino, nitro, sulfhydryl, alkylthio, imine, amide, phosphoryl, phosphonate, phosphine, carbonyl, carboxyl, carboxamide, anhydride, silyl, thioalkyl, alkylsulfonyl, arylsulfonyl, selenoalkyl, ketone, aldehyde, ester, heteroalkyl, nitrile, guanidine, amidine, acetal, ketal, amine oxide, aryl, heteroaryl, azide, aziridine, carbamate, epoxide, hydroxamic acid, imide, oxime, sulfonamide, thioamide, thiocarbamate, urea, thiourea, or -(CH2)
  • a and B are substituted or unsubstituted aryls, preferably substituted with one or more hydrophobic groups;
  • X represents N or CR;
  • R represents independently for each occurrence H, halogen, alkyl, alkenyl, alkynyl, hydroxyl, alkoxyl, silyloxy, amino, nitro, sulfhydryl, alkylthio, imine, amide, phosphoryl, phosphonate, phosphine, carbonyl, carboxyl, carboxamide, anhydride, silyl, thioalkyl, alkylsulfonyl, arylsulfonyl, selenoalkyl, ketone, aldehyde, ester, heteroalkyl, nitrile, guanidine, amidine, acetal, ketal, amine oxide, aryl, heteroaryl, azide, aziridine, carbamate, epoxide, hydroxamic acid, imide, oxime, sulfonamide, thioamide, thiocarbamate, urea, thiour
  • R 8 o represents independently for each occurrence aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and m is an integer in the range 0 to 8 inclusive.
  • the compounds of the present invention are represented by structure B and the attendant definitions, wherein X represents independently for each occurrence N.
  • the compounds of the present invention are represented by structure B and the attendant definitions, wherem R represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R 8 o-
  • R represents independently for each occurrence H, halogen, alkyl, alkenyl, alkynyl, hydroxyl, alkoxyl, silyloxy, amino, nitro, sulfhydryl, alkylthio, imine, amide, phosphoryl, phosphonate, phosphine, carbonyl, carboxyl, carboxamide, anhydride, silyl, thioalkyl, alkylsulfonyl, arylsulfonyl, selenoalkyl, ketone, aldehyde, ester, heteroalkyl, nitrile, guanidine, amidine, acetal, ketal, amine oxide, aryl, heteroaryl, azide, aziridine, carbamate, epoxide, hydroxamic acid, imide, oxime, sulfonamide, thioamide, thiocarbamate, urea, thiourea, or -(CH2)
  • R' represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, 2-aminoacyl, 3-aminoacyl, alkylC(0)alkyl, arylC(0)alkyl, sulfonyl, alkoxycarbonyl, alkylaminocarbonyl, or -(CH2) -R 8 o;
  • R" represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, 2-aminoacyl, 3-aminoacyl, alkylC(0)alkyl, arylC(0)alkyl, sulfonyl, alkoxycarbonyl, alkylaminocarbonyl, or -(CH2) m -R 8 o;
  • the compounds of the present invention are represented by structure C and the attendant definitions, wherein X represents independently for each occurrence CH 2 .
  • the compounds of the present invention are represented by structure C and the attendant definitions, wherein R represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R 8 o.
  • a compound of the present invention is represented by D:
  • X 1 is selected independently for each occurrence from the group consisting of F, CI, Br, and I and a substituted or unsubstituted benzene ring;
  • X 2 is selected from the group consisting of O or S.
  • R represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R 8 o;
  • R' represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R. 8 o;
  • the compounds of the present invention are represented by structure D and the attendant definitions, wherein X represents CI or I.
  • the compounds of the present invention are represented by structure D and the attendant definitions, wherein R represents independently for each occurrence H.
  • the compounds of the present invention are represented by stmcture D and the attendant definitions, wherein R' represents independently for each occurrence H.
  • the compounds of the present invention are represented by stmcture D and the attendant definitions, wherein X represents CI or I; R represents independently for each occurrence H; and R' represents independently for each occurrence H.
  • a compound of the present invention is represented by E:
  • R represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R 80 ;
  • R 80 represents independently for each occurrence aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and m is an integer in the range 0 to 8 inclusive.
  • the compounds of the present invention are represented by stmcture E and the attendant definitions, wherein X represents CI or I.
  • a compound of the present invention is represented by F:
  • R represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R 8 o;
  • R' represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R 8 o;
  • R 8 o represents independently for each occurrence aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and m is an integer in the range 0 to 8 inclusive.
  • the compounds of the present invention are represented by stmcture F and the attendant definitions, wherein R represents independently for each occurrence H.
  • the compounds of the present invention are represented by stmcture F and the attendant definitions, wherein R' represents independently for each occurrence alkyl. In certain embodiments, the compounds of the present invention are represented by stmcture F and the attendant definitions, wherein R represents independently for each occurrence H; and R' represents independently for each occurrence alkyl. In certain embodiments, a compound of the present invention is represented by G:
  • R represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R 8 o;
  • R' represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R. 8 o;
  • R 8 o represents independently for each occurrence aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and m is an integer in the range 0 to 8 inclusive.
  • the compounds of the present invention are represented by stmcture G and the attendant definitions, wherein R represents independently for each occurrence H.
  • the compounds of the present invention are represented by stmcture G and the attendant definitions, wherein R' represents independently for each occurrence alkyl.
  • the compounds of the present invention are represented by stmcture G and the attendant definitions, wherein R represents independently for each occurrence H; and R' represents independently for each occurrence alkyl.
  • a compound of the present invention is represented by H:
  • R represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R 8 o;
  • R 8 o represents independently for each occurrence aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and
  • m is an integer in the range 0 to 8 inclusive.
  • the compounds of the present invention are represented by stmcture H and the attendant definitions, wherein R represents independently for each occurrence H.
  • a compound of the present invention is represented by I:
  • R represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R 8 o;
  • R' represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R 8 o;
  • R 8 o represents independently for each occurrence aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and m is an integer in the range 0 to 8 inclusive.
  • the compounds of the present invention are represented by stmcture I and the attendant definitions, wherein R represents independently for each occurrence H.
  • R represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R o;
  • R' represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -R 8 o;
  • R 80 represents independently for each occurrence aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and m is an integer in the range 0 to 8 inclusive.
  • the compounds of the present invention are represented by stmcture J and the attendant definitions, wherein R represents independently for each occurrence H.
  • a compound of the present invention is represented by K:
  • R represents independently for each occurrence H, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2) m -K-8o;
  • R 8 o represents independently for each occurrence aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and m is an integer in the range 0 to 8 inclusive.
  • the compounds of the present invention are represented by stmcture K and the attendant definitions, wherein R represents independently for each occurrence H.
  • the compounds of the present invention are represented by stmcture K and the attendant definitions, wherein R' represents independently for each occurrence alkyl. In certain embodiments, the compounds of the present invention are represented by stmcture K and the attendant definitions, wherein R represents independently for each occurrence H; and R' represents independently for each occurrence alkyl.
  • the compounds of the present invention are represented by stmcture K and the attendant definitions, wherein R represents independently for each occurrence H; and R' represents independently for each occurrence methyl or ethyl.
  • the above-described compounds can be prepared according to methods known in the art, e.g., as described in "Comprehensive Organic Transformations: A Guide to Functional Group Preparations” by Richard C. Larock, VCH Publishers (1989) and “Advanced Organic Chemistry: Reactions, Mechanisms, and Stmcture” Fourth Edition, Jerry March, Wiley Interscience (1992). At least some of the steps of the synthesis can be identical to those used to prepare the chemical library from which some of the compounds of the invention were isolated.
  • the compounds of the invention can be purchased from companies, e.g., companies involved in custom synthesis of chemical compounds.
  • Exemplary companies which synthesize compounds, e.g., F16, are Interbioscreen Ltd., Moscow, Russia (the catalogue number for F16 is 2S-13965); Asinex Ltd., Moscow, Russia (the catalogue number for F16 is BAS 1027104); and ChemDiv, Inc., U.S. QR is available from Aldrich-Sigma, under catalog no. 20,131-6. Assays for testing the activity of compounds
  • Cell proliferation can also be determined by counting the cells at different times. Viable cells can be counted by staining the cells with a specific dye, e.g., Trypan Blue, according to methods well known in the art. Other methods include measuring the incorporation of a labeled molecule into DNA or RNA or protein of cells. For example, cell proliferation is often by measured by 3 H thymidine or 5-bromodeoxyuridine incorporation assays, also well known in the art.
  • the effect of a compound on cell differentiation can be determined by visualization of the cells after having been contacted with the compound, preferably by comparison with cells which have not been contacted with the compound.
  • the differentiation of certain cells is visible by the naked eye (e.g., that of 3T3L1 cells), whereas that of other cells may require the use of a microscope.
  • Specific dyes can also be used to evaluate the state of differentiation of cells.
  • Cell differentiation can also be monitored by measuring the expression level of certain genes, whose expression is known to vary during differentiation of the cells.
  • Cells that can be used for testing compounds of the invention include cell lines and primary cell cultures.
  • Acute Type Culture Collection ATCC
  • Primary cell cultures can be established from biopsies obtained from subjects. For example, primary tissue cultures of cells over-expressing an activated form of Neu can be prepared from biopsies of subjects having breast cancer.
  • Cell lines over-expressing a gene, e.g., a proto-oncogene can be prepared by transient, or preferably, stable transfection of cells with an expression plasmid containing the gene. Transfection methods are well known in the art and are also described in the examples. Nucleic acids for transforming cells, e.g., proto-oncogenes are also available in the art.
  • MG 1361 is a breast carcinoma cell line obtained from the MMTV-neu transgenic mouse (Sacco et al., Breast Cancer Res. Treat, 47:171-180 (1998)).
  • the present invention also provides methods of testing a compound (e.g., the candidate dmg) for its antiproliferative effect; its effect on cell differentiation; its induction of cell death; and/or its toxicity on normal or wild-type cells in a transgenic animal, e.g., mouse.
  • Transgenic mice can be produced that express a transforming agent (e.g., a growth factor receptor) under the control of a promoter, e.g., a tissue specific promoter. Such mice develop carcinomas that have genetic and pathological features that closely resemble human cancers.
  • the ability of the compound to inhibit tumor formation or growth can then be ascertained.
  • the effect on tumors is monitored by determining the tumor size and/or weight.
  • the compounds can be administered by a variety of ways including orally, subcutaneously, or intraperitoneally. Generally, at least two groups of animals are used in the assay, with at least one group being a control group, e.g., receiving the vehicle without the compound.
  • mice transgenic for an oncogene are described in the following publications. MMTV-neu transgenic mice are described in Muller et al. (1988) Cell 54: 105. A neu transgenic mouse in which the Neu coding region is under the control of the normal neu promoter is described in Weinstein et al. (2000) Mol. Med. 6:4. A transgenic mouse expressing activated ras is described, e.g., in Kohl, et al., Nature Medicine, vol. 1, No. 8 (Aug. 1995). A myc transgenic mouse is described in U.S. Patent No. 5,925,803, by Leder et al., which also describes methods for making mice that are transgenic for any one of a variety of oncogenes.
  • ATCC American Type Culture Collection
  • the compounds of the invention can be used to normalize, e.g., inhibit or block the proliferation of cells and/or modulate the differentiation of cells.
  • the compounds can also be used to kill target cells, e.g., by inducing apoptosis in the cells.
  • target cells The cells whose proliferation or differentiation is to be normalized or which are to be killed are referred herein as "target cells.”
  • a target cell can essentially be any cell whose proliferation is to be inhibited or its differentiation modulated or which is to be killed.
  • target cells can be cells which are defective, e.g., non-responsive to, normal cell proliferation control mechanisms.
  • a target cell can be not only a cell expressing the Neu or Ras or Myc oncogene or ⁇ -catenin (as described in the Examples), but it can also be a cell expressing another oncogene.
  • a compound which has an antiproliferative effect against a cell transformed with a Neu oncogene can also have an antiproliferative effect against a cell having another defect, e.g., the presence of an oncogene.
  • the cell can be "cured" not only by effecting that particular molecule, but by compensating for the effect of the defect in the particular molecule by effecting upstream or downstream molecules in the same signal transduction pathway, or in a parallel signal transduction pathway.
  • the compounds of the invention are expected to be effective against a broad range of target cells, and not only target cells transformed with an oncogene.
  • at least some of the compounds of the invention may be effective against cells which proliferate and/or differentiate normally, i.e., wild-type cells. For example, certain compounds could be used to arrest cell proliferation.
  • the efficacy of the compounds of the invention against a broad range of target cells allows for broad applications for these compounds.
  • the compounds of the invention are effective at very low doses.
  • F16 is effective at lOnM (see, e.g., Figure 3).
  • these compounds were shown not to affect certain other cells. For example, most compounds did not significantly inhibit proliferation of the counter-part non-transformed cells and some of the compounds only affected cells transformed with certain oncogenes and not others.
  • the invention provides methods for treating cancer, e.g., cancers that are caused by, or associated with, expression of an oncogene or over-expression of a proto-oncogene, e.g., the Neu proto-oncogene.
  • cancers e.g., cancers that are caused by, or associated with, expression of an oncogene or over-expression of a proto-oncogene, e.g., the Neu proto-oncogene.
  • An exemplary cancer that can be treated is breast cancer, in particular, forms of breast cancers which are associated with an over- expression of the Neu proto-oncogene.
  • Amplification and/or overexpression of the human erbB2 gene correlates with a poor prognosis in breast and ovarian cancers, in particular, carcinomas.
  • Slamon et al. Science 235:177-82 (1987); Slamon et al., Science 244:707-12 (1989).
  • Overexpression of erbB2 has been correlated with other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon and bladder. Accordingly, in U.S. Pat. No. 4,968,603, Slamon et al. describe and claim various diagnostic assays for determining erbB2 gene amplification or expression in tumor cells. Slamon et al.
  • the compounds of the invention have been isolated based on their ability to inhibit growth of cells that are transformed with an activated form of the Neu proto- oncogene, in view of the similarity of the Neu proto-oncogene with other members of the family of EGF receptors, the compounds of the invention are likely to similarly inhibit the proliferation of cells transformed with other members of the EGF receptor family, such as Neu-erb2 -related genes. Accordingly, the compounds of the invention can be used for treating diseases, in particular, malignancies, that are associated with the presence of such related genes. The effectiveness of the compounds in treating these other diseases can be tested using appropriate cell lines or animal models, as further described herein.
  • a further related gene of the Neu proto-oncogene is erbB3 (or HER3), which encodes the ErbB-3 receptor (pl80.sup.HER3).
  • This receptor has been described, e.g., in U.S. Pat. Nos. 5,183,884 and 5,480,968; Kraus et al., PNAS USA 86:9193-97 (1989); EP Patent Application No. 444,961 Al; Kraus et al., PNAS USA 90:2900-04 (1993).
  • the invention further provides methods for treating proliferative diseases which are associated with the presence of an activated form of the Ras proto-oncogene.
  • certain compounds that were identified as having an antiproliferative activity against cells expressing an activated form of the Neu proto-oncogene are also effective against cells expressing an activated form of the Ras proto-oncogene.
  • the Ras protein is part of a signaling pathway that links cell surface growth factor receptors to nuclear signals initiating cellular proliferation.
  • Biological and biochemical studies of Ras action indicate that Ras functions like a G-regulatory protein.
  • Ras In the inactive state, Ras is bound to GDP.
  • Ras Upon growth factor receptor activation, Ras is induced to exchange GDP for GTP and undergoes a conformational change.
  • the GTP-bound form of Ras propagates the growth stimulatory signal until the signal is terminated by the intrinsic GTPase activity of Ras, which returns the protein to its inactive GDP bound form (D. R. Lowy and D. M. Willumsen, Ann. Rev. Biochem. 62:851-891 (1993)).
  • Activation of Ras leads to activation of multiple intracellular signal transduction pathways, including the MAP Kinase pathway and the Rho/Rac pathway (Joneson et al., Science 271 :810-812).
  • H-ras is an abbreviation for Harvey-ras.
  • K4A-ras and K4B-ras are abbreviations for the Kirsten splice variants of ras that contain the 4 A and 4B exons, respectively.
  • Mutated ras genes are found in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias. The protein products of these genes are defective in their GTPase activity and constitutively transmit a growth stimulatory signal.
  • Cancers associated with activated Ras that can be treated include epithelial cancers, e.g., prostate cancer, lung cancer, breast cancer, colorectal cancer, and pancreatic cancer.
  • Activated Myc oncogenes have been shown to cause testicular, breast, lymphocytic (T and B cell) cancers, e.g., pre-B cell lymphomas, Burtkitt's lymphoma as well as cancers of mast cell origin (Leder et al. (1986) Cell 45: 485; Suda et al. (1987) EMBO J. 20: 4055; and Chung et al. (1986) PNAS 83:7918). Accordingly, the compounds of the invention can be used for treating such diseases.
  • proliferative diseases which may be treated with the compounds of the invention include: the benign proliferative disorder neurofibromatosis, or tumors in which ras is activated due to mutation or overexpression of tyrosine kinase oncogenes (e.g., neu, src, abl, lck, lyn, fyn).
  • tyrosine kinase oncogenes e.g., neu, src, abl, lck, lyn, fyn.
  • a preferred therapeutic effect provided by the instant composition is the treatment of cancer and specifically the inhibition of cancerous tumor growth and/or the regression of cancerous tumors.
  • the cancer can result from the presence of an oncogene or from another cause.
  • Cancers, which are treatable in accordance with the invention described herein include cancers of the brain, breast, colon, genitourinary tract, prostate, skin, lymphatic system, pancreas (e.g., pancreatic carcinoma such as, for example, exocrine pancreatic carcinoma), rectum, stomach, larynx, ovary, bladder, liver and lung.
  • cancers include histiocytic lymphoma, lung adenocarcinoma, pancreatic carcinoma, colo-rectal carcinoma, small cell lung cancers, bladder cancers, head and neck cancers, acute and chronic leukemias, melanomas, neurological tumor, myeloid leukemias (for example, acute myelogenous leukemia), sarcomas, thyroid follicular cancer, and myelodysplastic syndrome.
  • a growth factor e.g., platelet derived growth factor (PDGF), fibroblast derived growth factor (FGF), epidermal derived growth factor (EGF) and vascular endothelial growth factor (VEGF).
  • PDGF platelet derived growth factor
  • FGF fibroblast derived growth factor
  • EGF epidermal derived growth factor
  • VEGF vascular endothelial growth factor
  • the compounds of the invention can also be used for treating smooth muscle cell hyper-proliferation, at least in part since PDGF is considered to be a principal growth- regulatory molecule responsible for smooth muscle cell proliferation.
  • One smooth muscle disorder is atherosclerosis, which is a disease characterized by focal thickening of the inner portion of the artery wall, predisposing an individual to myocardial infarction (heart attack), cerebral infarction (stroke), hypertension (high blood pressure) and gangrene of the extremities.
  • atherosclerosis is a disease characterized by focal thickening of the inner portion of the artery wall, predisposing an individual to myocardial infarction (heart attack), cerebral infarction (stroke), hypertension (high blood pressure) and gangrene of the extremities.
  • lesions of atherosclerosis are surrounded by large amounts of lipid-laden macrophages, varying numbers of lymphocytes and large amounts of connective tissue.
  • PDGF has been found in numerous cells in such lesions, and it is believed that PDGF plays a critical role in the atherosclerosis disease process.
  • Other smooth muscle diseases include diabetic vascular pathologies.
  • Another smooth muscle cell disease that can be treated according to the invention is restenosis. This disease is characterized by the regrowth of smooth muscle cells into the lumen of blood vessels following angioplasty or other arterial damage, is a frequent and recurring problem in the long term success of angioplasty.
  • composition of the instant invention is also useful in the prevention of restenosis after percutaneous transluminal coronary angioplasty by inhibiting neointimal formation (C. Indolfi et al. Nature medicine, 1 :541-545(1995)).
  • the compounds of the invention are used for treating rheumatoid arthritis (R.A.).
  • Synovial tissues of RA patients express high levels of FGF and PDGF compared with synovial tissues of osteoarthritis patients, a non invasive joint disease (Sano et al., J. Cell. Biol. 110:1417-1426, 1990). These data are consistent with the theory that PDGF and FGF play a role in generating an invasive tumor-like behavior in arthritic joints of RA synovial connective tissues (Sano et al., J. Clin. Invest. 91 :553-565 (1993)).
  • Both FGF and VEGF are potent angiogenic factors which induce formation of new capillary blood vessels. Accordingly, the compounds of the invention may be useful in inhibiting vascularization, e.g., in tumors.
  • the instant composition may also be useful in the treatment and prevention of polycystic kidney disease (D. L. Schaffner et al. American Journal of Pathology, 142: 1051- 1060 (1993) and B. Cowley, Jr. et al. FASEB Journal, 2:A3160 (1988)).
  • the pharmaceutical preparations of the invention are intended for the treatment of hyperplastic epidermal conditions, such as keratosis, as well as for the treatment of neoplastic epidermal conditions such as those characterized by a high proliferation rate for various skin cancers, as for example basal cell carcinoma or squamous cell carcinoma.
  • the subject method can also be used in the treatment of autoimmune diseases affecting the skin, in particular, of dermatological diseases involving morbid proliferation and/or keratinization of the epidermis, as for example, caused by psoriasis or atopic dermatosis.
  • psoriasis squamous cell carcinoma, keratoacanthoma and actinic keratosis
  • psoriasis which is characterized by scaly, red, elevated plaques on the skin
  • the keratinocytes are known to proliferate much more rapidly than normal and to differentiate less completely.
  • the preparations of the present invention are suitable for the treatment of dermatological ailments linked to keratinization disorders causing abnormal proliferation of skin cells, which disorders may be marked by either inflammatory or noninflammatory components.
  • therapeutic preparations of a compound of the invention can be used to treat varying forms of psoriasis, be they cutaneous, mucosal or ungual.
  • Psoriasis as described above, is typically characterized by epidermal keratinocytes which display marked proliferative activation and differentiation along a "regenerative" pathway.
  • Treatment with an antiproliferative embodiment of the subject method can be used to reverse the pathological epidermal activition and can provide a basis for sustained remission of the disease.
  • a variety of other keratotic lesions are also candidates for treatment with the subject antiproliferative preparations.
  • Actinic keratoses are superficial inflammatory premalignant tumors arising on sun-exposed and irradiated skin. The lesions are erythematous to brown with variable scaling. Current therapies include excisional and cryosurgery. These treatments are painful, however, and often produce cosmetically unacceptable scarring. Accordingly, treatment of keratosis, such as actinic keratosis, can include application, preferably topical, of a composition comprising one or more compounds of the invention in amounts sufficient to inhibit hyperproliferation of epidermal/epidermoid cells of the lesion.
  • Acne represents yet another dermatologic ailment which may be treated with an antiproliferative embodiment of the subject method.
  • Acne vulgaris for instance, is a multifactorial disease most commonly occurring in teenagers and young adults, and is characterized by the appearance of inflammatory and noninflammatory lesions on the face and upper trunk.
  • the basic defect which gives rise to acne vulgaris is hypercomification of the duct of a hyperactive sebaceous gland. Hypercomification blocks the normal mobility of skin and follicle microorganisms, and in so doing, stimulates the release of lipases by Propinobacterium acnes and Staphylococcus epidermidis bacteria and Pitrosporum ovale, a yeast.
  • Treatment with an antiproliferative therapeutic may be useful for preventing the transitional features of the ducts, e.g. hypercomification, which lead to lesion formation.
  • the subject treatment may further include, for example, antibiotics, retinoids and antiandrogens.
  • the present invention also provides a method for treating various forms of dermatitis.
  • Dermatitis is a descriptive term referring to poorly demarcated lesions which are either pmritic, erythematous, scaley, blistered, weeping, fissured or cmsted. These lesions arise from any of a wide variety of causes. The most common types of dermatitis are atopic, contact and diaper dermatitis.
  • seborrheic dermatitis is a chronic, usually pmritic, dermatitis with erythema, dry, moist, or greasy scaling, and yellow cmsted patches on various areas, especially the scalp, with exfoliation of an excessive amount of dry scales stasis dermatitis, an often chronic, usually eczematous dermatitis.
  • Actinic dermatitis is dermatitis that due to exposure to actinic radiation such as that from the sun, ultraviolet waves or x- or gamma-radiation.
  • the subject therapeutic preparations can be used in the treatment and/or prevention of certain symptoms of dermatitis caused by unwanted proliferation of epithelial cells.
  • Such therapies for these various forms of dermatitis can also include topical and systemic corticosteroids, antipuritics, and antibiotics.
  • antiproliferative preparations of therapeutics can be used to inhibit lens epithelial cell proliferation to prevent post-operative complications of extracapsular cataract extraction.
  • Cataract is an intractable eye disease and various studies on a treatment of cataract have been made. But at present, the treatment of cataract is attained by surgical operations. Cataract surgery has been applied for a long time and various operative methods have been examined. Extracapsular lens extraction has become the method of choice for removing cataracts. The major medical advantages of this technique over intracapsular extraction are lower incidence of aphakic cystoid macular edema and retinal detachment. Extracapsular extraction is also required for implantation of posterior chamber type intraocular lenses which are now considered to be the lenses of choice in most cases.
  • a disadvantage of extracapsular cataract extraction is the high incidence of posterior lens capsule opacification, often called after-cataract, which can occur in up to 50% of cases within three years after surgery.
  • After-cataract is caused by proliferation of equatorial and anterior capsule lens epithelial cells which remain after extracapsular lens extraction. These cells proliferate to cause Sommerling rings, and along with fibroblasts which also deposit and occur on the posterior capsule, cause opacification of the posterior capsule, which interferes with vision. Prevention of after-cataract would be preferable to treatment.
  • the subject method provides a means for inhibiting proliferation of the remaining lens epithelial cells. For example, such cells can be induced to remain quiescent by instilling a solution containing an antiproliferative therapeutic preparation into the anterior chamber of the eye after lens removal.
  • the solution can be osmotically balanced to provide minimal effective dosage when instilled into the anterior chamber of the eye, thereby inhibiting subcapsular epithelial growth with some specificity.
  • the subject method can also be used in the treatment of corneopathies marked by comeal epithelial cell proliferation, as for example in ocular epithelial disorders such as epithelial downgrowth or squamous cell carcinomas of the ocular surface.
  • the subject method can be used in conjunction with various periodontal procedures in which control of epithelial cell proliferation in and around periodontal tissue is desired.
  • Yet another aspect of the present invention relates to the use of therapeutic preparations to control hair growth.
  • certain therapeutics e.g., antiproliferative forms
  • the present method can be used in the treatment of trichosis characterized by abnormally rapid or dense growth of hair, e.g. hypertrichosis.
  • the compounds of the invention can be used to manage hirsutism, a disorder marked by abnormal hairiness.
  • the subject method can also provide a process for extending the duration of depilation.
  • compounds of the invention will often be cytostatic to epithelial cells, rather than cytotoxic, such agents can be used to protect hair follicle cells from cytotoxic agents which require progression into S-phase of the cell-cycle for efficacy, e.g. radiation-induced death.
  • Treatment by the subject method can provide protection by causing the hair follicle cells to become quiescent, e.g., by inhibiting the cells from entering S phase, and thereby preventing the follicle cells from undergoing mitotic catastrophe or programmed cell death.
  • compounds of the invention can be used for patients undergoing chemo- or radiation-therapies which ordinarily result in hair loss. By inhibiting cell-cycle progression during such therapies, the subject treatment can protect hair follicle cells from death which might otherwise result from activation of cell death programs.
  • the subject method can also be used in the treatment of folliculitis, such as folliculitis decalvans, folliculitis ulerythematosa reticulata or keloid folliculitis.
  • folliculitis such as folliculitis decalvans, folliculitis ulerythematosa reticulata or keloid folliculitis.
  • a cosmetic prepration of a compound of the invention can be applied topically in the treatment of pseudofolliculitis, a chronic disorder occurring most often in the submandibular region of the neck and associated with shaving, the characteristic lesions of which are erythematous papules and pustules containing buried hairs.
  • the compounds of the invention can also be used to inhibit proliferation and/or kill cells having a high ⁇ m.
  • Cells having an abnormally high ⁇ m include cancer cells, e.g., human breast adenocarcinomas (see, e.g., Davis et al. (1985) J. Biol. Chem. 260:13844; Summerhayes et al. (1982) PNAS 79:5292; Modica-Napolitano et al. (1998) Cancer Res. 58:71; and Modica-Napolitano et al. (1990) Cancer Res. 50:7876; and Nadakavukaren et al. (1985) Cancer Res. 45: 6093).
  • a compound of the invention is administered to a subject having cancer, e.g., in a tumor of a subject, and the tumor is subjected to high-intensity light to increase the anti-proliferative and/or cytotoxic effect of the compounds.
  • the compounds of the invention can be used as anti-fungal agents to treat fungal infections on animals, e.g., humans.
  • the compounds can also be used for stopping fungus growth on objects, e.g., mold growth on shower curtains.
  • Methods of administration of the compounds of the invention generally comprises administering to a subject in need thereof, a pharmaceutically effective amount of a compound.
  • the compounds of the invention can be administered in a "growth inhibitory amount," i.e., an amount of the compound which is pharmaceutically effective to inhibit or decrease proliferation of target cells.
  • Toxicity and therapeutic efficacy of the compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Reagents which exhibit large therapeutic indices are preferred. While reagents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such reagents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such reagents lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • Such information can be used to more accurately determine useful doses in humans.
  • some compounds of the invention are effective at concentrations of 1 OnM, 1 OOnM, or 1 ⁇ M. Based on these numbers, it is possible to derive an appropriate dosage for administration to subjects.
  • a water soluble taste masking material such as hydroxypropylmethyl-cellulose or hydroxypropylcellulose, or a time delay material such as ethyl cellulose, cellulose acetate buryrate may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
  • preservatives for example ethyl, or n-propyl p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • flavoring agents such as sucrose, saccharin or aspartame.
  • sweetening agents such as sucrose, saccharin or aspartame.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.
  • These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
  • sweetening agents for example glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
  • compositions may be in the form of a sterile injectable aqueous solutions.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • the sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion where the active ingredient is dissolved in the oily phase.
  • the active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution then introduced into a water and glycerol mixture and processed to form a microemulation.
  • the injectable solutions or microemulsions may be introduced into a patient's bloodstream by local bolus injection. Alternatively, it may be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating concentration of the instant compound.
  • a continuous intravenous delivery device may be utilized.
  • An example of such a device is the Deltec CADD-PLUSTM model 5400 intravenous pump.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3 -butane diol.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Compounds of the invention may also be administered in the form of a suppositories for rectal administration of the dmg.
  • compositions can be prepared by mixing the dmg with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the dmg.
  • suitable non-irritating excipient include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
  • creams, ointments, jellies, solutions or suspensions, etc., containing the compound of the invention are employed.
  • topical application shall include mouth washes and gargles.
  • the compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • the compounds identified by the instant method may also be co-administered with other well known therapeutic agents that are selected for their particular usefulness against the condition that is being treated.
  • the instant compounds may be useful in combination with known anti-cancer and cytotoxic agents.
  • the instant compounds may be useful in combination with agents that are effective in the treatment and prevention of neurofibromatosis, restinosis, polycystic kidney disease, infections of hepatitis delta or HPV and related vimses and fungal infections.
  • the instant compounds may also be useful in combination with other inhibitors of parts of the signaling pathway that links cell surface growth factor receptors to nuclear signals initiating cellular proliferation.
  • chemotherapeutic agent examples include: alkylating agents, antimetabolites, natural products and their derivatives, hormones and steroids (including synthetic analogs), and synthetics. Examples of compounds within these classes are given below.
  • Alkylating agents including nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes: Uracil mustard, Chlormethine, Cyclophosphamide (CytoxanTM), Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylene-melamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, dacarbazine, and Temozolomide.
  • Antimnetabolites including folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors: Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6- Mercaptopurine, 6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.
  • Natural products and their derivatives including vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins: Vinblastine, Vincristine, Vindesine, Bleomycin, Dactinomycin, Daunorubicin, Doxombicin, Epimbicin, Idarubicin, paclitaxel (paclitaxel is commercially available as Taxol.RTM. and is described in more detail below in the subsection entitled "Microtubule Affecting Agents"), Mithramycin, Deoxycoformycin, Mitomycin-C, L-Asparaginase, Interferons (especially IFN-a), Etoposide, and Teniposide.
  • Hormones and steroids include synthetic analogs: 17.alpha.-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Megestrolacetate, Tamoxifen, Methylprednisolone, Methyltestosterone, Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, Zoladex.
  • Synthetics including inorganic complexes such as platinum coordination complexes: Cisplatin, Carboplatin, Hydroxyurea, Amsacrine, Procarbazine, Mitotane, Mitoxantrone, Levamisole, and Hexamethylmelamine.
  • combination products employ the combinations of this invention within the dosage range described below and the other pharmaceutically active agent(s) within its approved dosage range.
  • Combinations of the instant invention may also be used sequentially with known pharmaceutically acceptable agent(s) when a multiple combination formulation is inappropriate.
  • composition according to this invention When a composition according to this invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, and response of the individual patient, as well as the severity of the patient's symptoms. Additional uses for the compounds of the invention
  • At least two compounds create a fluorescence in cells that are contacted with it and which are cells that will be killed by the compound.
  • at least some compounds of the invention can be used as markers of cells that are susceptible of being killed or cell cycle arrested by the compounds or of cells which are being killed or cell cycle arrested.
  • cells that have been killed may be discarded by the body, it may be difficult to assess exactly the effect of the compound.
  • compounds which inhibit cell proliferation these are also particularly useful, since it may not otherwise be possible to identify cells which have been cell cycle arrested. Based on the stmcture of the compounds identified, in particular, the structure of
  • kits All the essential materials and reagents required for administering the compounds of the invention may be assembled together in a kit.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the compound may be provided in combination with one or more other d gs, e.g., chemo- or radiotherapeutic agent.
  • d gs e.g., chemo- or radiotherapeutic agent.
  • chemo- or radiotherapeutic agent normally will be a separate formulation, but may be formulated into a single pharmaceutically acceptable composition.
  • the container means may itself be geared for administration, such as an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, or injected into an animal, or even applied to and mixed with the other components of the kit.
  • the compositions of these kits also may be provided in dried or lyophilized forms.
  • kits of the invention may also include an instruction sheet defining administration of the agent and, e.g., explaining how the agent will decrease proliferation of cells.
  • kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • the kits of the invention also may comprise, or be packaged with a separate instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal.
  • a separate instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal.
  • Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
  • Other instmmentation includes devices that permit the reading or monitoring of compound levels or reactions in vitro.
  • EpH4 cells which are non-transformed mouse mammary epithelial cells, which grow in a monolayer exhibiting the characteristic "cobble stone" shape of epithelial cells were chosen for stable transfection with the Neu oncogene.
  • EpH4 cells were described by Reichmann (1992) Cell 71 :1103 as a subclone of a mixed epithelial /mesenchymal polyclonal line originally reported in Reichman et al. (1989) J. Cell Biol. 108:1 127.
  • EpH4 is an acronym for epithelial (Ep) hygromycin (H) clone 4 (4).
  • EpH4 cells were grown in Dulbecco's modified Eagle's medium (DMEM), 5 % FBS at 37°C/ 5% C0 2 .
  • DMEM Dulbecco's modified Eagle's medium
  • EpH4 cells stably expressing the full length rat Neu coding sequence were prepared as follows.
  • the Neu transforming oncogene (Neu ⁇ ) cDNA GenBank Accession
  • NoX03362 was subcloned into the plasmid pRETRO-OFF (Clontech) as follows.
  • a Sall/Xhol/Hindlll/Notl adapter was introduced into pBluescript (Stratagene).
  • the Hindlll/Sall fragment containing full length Neu ⁇ cDNA was excised from a vector, which itself was derived from pSV2neuNT (Bergman (1986) Nature 3190:226) and inserted into corresponding sites in p-Bluescript.
  • EpH4 cells were stably transfected with the empty pRETRO-OFF vector or with the pRETRO-OFF vector in which the Neu ⁇ cDNA was inserted, using Fugene Reagent according to manufacturer's protocol (Boehringer Mannheim). Stable clones were isolated by selection on puromycin at 1.2 ⁇ g/ml. Several stably transfected clones were obtained. One clone having stably integrated the vector, and referred to as "EpH4-EV" and two clones having stably integrated the vector with the Neu ⁇ cDNA, referred to as "EpH4-A6" and "EpH4-E8,” were used to identify anti-proliferative compounds.
  • EpH4 cells two of the major signaling pathways mediating the effects of activated Neu, the MAP kinase and the PI3K/PKB pathways, were examined. Accordingly, the protein level of pPKB, p44-42 MAPK, Neu, and ⁇ -actin (as a control) proteins were determined in Neu- transformed and control cells, as follows.
  • EpH4, EpH4-A6 and EpH4-A8 were incubated in serum-free medium for 5 hrs and subsequently lysed in lysis buffer with protease inhibitors (40 mM HEPES, 150 mM NaCl, 10 mM sodium pyrophosphate, 1% NP40 ,10mM NaF, 2mM EDTA, 10 g/mL aprotinin, 10 M leupeptin, 10 g/mL pepstatin, 10 M EP475, 1 M PMSF, 10 M Na3V ⁇ 4.). The lysate was spun at 140,000 x g for 30 min to separate the insoluble material. Protein concentration was determined on the supernatant using Bradford reagent (Bio-Rad).
  • the results also indicate that the Neu protein is expressed in EpH4-A6 and -A8 and is phosphorylated, as expected, but is not detectable in EpH4 cells or in EpH4-EV cells. It was also shown that these Neu- overexpressing cells form fast growing tumors when injected into nude mice, whereas as the non-expressing cells did not, further confirming that they are transformed cells.
  • Neu is capable of active signaling in the EpH4 cells, providing a good experimental system to utilize in a high-throughput screen for inhibitors of Neu-mediated growth.
  • Example 2 Identification of compounds which inhibit proliferation of activated Neu- overexpressing cells This example describes the identification of six compounds which inhibit proliferation of activated Neu-tranformed EpH4 cells.
  • cytoblot assay A modified version of a protocol previously described was used for the cytoblot assay (Stockwell et al. (1999) Chem Biol 6(2), 71-83). Briefly, for this cytoblot assay, 2,000 control (EpH4-EV) and Neu overexpressing (EpH4-A6) cells were seeded in 40 ⁇ L of growing medium in duplicate in each well of a white 352-well plate (Costar). 16,000 compounds from a chemical library (Chembridge) were transferred to each well to a final concentration of 10-15 ⁇ M. Plates were incubated at 37°C with 5% C0 2 for 36 hours. BrdU was then added to a final concentration of 20 ⁇ M and the cells were incubated at
  • F16 was able to partially inhibit BrdU inco ⁇ oration in EpH4-A6 cells at a concentration as low as 10 and 100 nM.
  • Two compounds that were found to be active at about 10 ⁇ M are set forth in Figures 2G and H, and their antiproliferative effect is shown in Figure 4. These two compounds are referred to as L4 (ID 121113); B15 (ID217496) and QR.
  • Figures 2 A-H also show proton NMR spectra of each of the molecules that confirmed their identity.
  • Example 3 Dose-response curve of the antiproliferative effect of the compounds
  • Example 4 Effect of Neu-inhibiting small molecules on cell lines transformed bv other oncogenes The effect of these six compounds (E20 (ID 141525); F12 (ID 120590); F16 (ID 274873); H10 (ID 120670); J6 (ID 120856); and N12 (ID 215015)), as well as that of the L4 and B15 compounds, was examined on neu, c-myc or v-Ha-ras initiated tumor cell lines.
  • the compounds were incubated at lO ⁇ M concentration with tumor cell lines expressing Neu (nNeu and Naf), Ras (AC816 and SH1,1), or Myc oncogene (16MB9a and 13MAla) or with EpH4-A6 or EpH4-EV cells as controls.
  • tumor cell lines expressing Neu (nNeu and Naf), Ras (AC816 and SH1,1), or Myc oncogene (16MB9a and 13MAla) or with EpH4-A6 or EpH4-EV cells as controls.
  • These tumor cell lines are described in Guy et al. (1996) J. Biol. Chem. 271 :7673; Muller et al. (1988) Cell 54: 105; Sinn et al. (1987) Cell 49:465 (SH1.1 and AC816); Stewart et al. (1984) Cell 38:627; and Leder et al. (1986) Cell 45:485, respectively.
  • the results, which are shown in Figure 4 show that the 6 compounds were also active on Neu and
  • This Example shows the effect of F16 on the proliferation of several human and mouse cell lines, and in particular, that the strongest anti-proliferative effect of FI 6 is on breast tumor lines.
  • the percentage of cells in the different phases of the cell cycle was assessed by measuring BrdU inco ⁇ oration into the DNA of the cells.
  • BrdU inco ⁇ oration into DNA of untreated or F16 treated (3 ⁇ M/ 30-36 hr) EpH-EV and EpH-A6 cells was assessed by FACS as follows. After F16 or mock treatment, the cells were pulsed with BrdU (20 ⁇ M) for 1 hour and then fixed in 70% ethanol at -20°C overnight. Next morning cells were washed twice with 1 x PBS and the DNA was denatured by incubation of the cells in 2N HCl/0.5% Triton X-100. The cells were then washed twice in 0.1M Na 2 B 2 0 7 , and once in PBS.
  • the cells were resuspended in PBS, incubated with 20 ⁇ L of anti-BrdU/FITC(Becton Dickinson) for 1 hour and subsequently washed with 1 x PBS and resuspended in PBS.
  • RNA was digested with RNaseA (1 ⁇ L of 10 mg/mL stock) at 37°C for 30 minutes.
  • the cells were diluted with 1 mL of 5 ⁇ g/mL propidium iodide/PBS and incubated on ice for 15 minutes.
  • the labeled cells were analyzed using the FACSCalibur (Becton Dickinson).
  • Figure 5 shows a representative result of these studies. These studies showed that F16 causes a dramatic decrease in the percentage of cells in the S phase of the cell cycle, and an increase in the percentage of cells in Gl phase. These results are in agreement with those obtained in the cytoblot screen assay (based on BrdU inco ⁇ oration).
  • Cytochrome c release was measured by immunofluorescence as follows. Untreated or F16-treated A6 cells were washed and fixed with ice-cold 3.7% paraformaldehyde at RT, permeabilized for with ice-cold 0.01% (v/v) Nonidet P-40 for 20 min and incubated with 0.5% BSA/PB. Immunodetection was performed with monoclonal anti-cytochrome c antibody (PharMingen) and secondary anti- mouse-TRITC conjugated antibody. Cells were photographed under Axioskop using a Spot Camera (Diagnostic Instruments).
  • Example 7 Effect of F16 in vivo As a first step towards evaluating the potential effect of F16 in vivo, pharmacokinetics studies with F16 were performed in nude mice.
  • mice were injected intraperitoneally with 25 mg/kg F16 and two mice were injected intraperitoneally with 12.5 mg/kg of F16.
  • the level of F16 in the semm of these mice was determined at 15 minutes, 30 minutes, 1 hour, 3 1/2 hours and 7 hours after the injection of F16, as follows. 40-50 ⁇ L of blood was obtained from the tail vein of the mice and placed into tubes incubated in ice and containing Heparin to a final concentration of 20U/mL of blood. Samples were centrifuged at 6,000 ⁇ m for 3 minutes in the cold room. Semm was transfered to a clean tube and stored at -70°C until further processing.
  • Fig. 8 and Table 4 shows the weight of the tumors of the control and F16 treated mice. These results indicate that a 2-3 fold difference in tumor size was observed in the F16 treated group relative to the control mice. The experiment was repeated with a second group of 20 mice, and similar results were obtained.
  • mice 2 10 mice were included in each group, but only nine F16 treated mice showed tumors.
  • mice The total weight of the mice was also monitored every other day during the course of the treatment with F 16. Placebo mice had an average total body weight gain of about 3.2%, whereas F16 treated mice had an average total body weight loss of 3.3%, relative to their weight at the beginning of the experiment.
  • F16 is a fluorescent compound with a maximum abso ⁇ tion of approximately 420 nm, and a maximum emission of approximately 520nm, which allows for visualization of its fluorescence signal. This property of F16 was used to determine the fate of the compound once inside the cell.
  • F16 used in this Example and in Examples 5-7 was obtained from Interbioscreen Ltd., Moscow, Russia (catalogue number 2S-13965) and from Asinex Ltd., Moscow, Russia (catalogue number BAS 1027104). The following cell lines were used in this Example: EpH4-A6 (Neu ⁇ ); EpH4-EV;
  • the cells were grown on 60 mm plates and incubated with medium alone or medium containing 3 ⁇ M FI 6 for 24 hours at 37°C.
  • the fluorescence signal of F16 was visualized under inverted microscope Axiovert (Zeiss) using a standard fluorescein filter set. Cells were photographed using a digital camera Dl (Nikon).
  • EpH4-EV and EpH4-A6 cells were seeded in 6 well chamber slides. Next morning, cells were left untreated or treated with F16 at 3 ⁇ M for 2 hours. Cells were incubated with Hoescht stain for 30 minutes and then visualized under the microscope. Hoescht stain specifically stains the nuclei of cells. A mitochondrial specific stain, Mitotracker Red (Molecular Probes), was added to the medium to stain mitochondria, and Lysotracker Red to stain lysosomes and acidic compartments, according to manufacturer procedure. Hoescht stain was also included in the medium (together with the other stains).
  • Mitotracker Red Molecular Probes
  • TMRM tetramethylrhodamin methyl ester
  • TMRM mitochondrial membrane potential of various mouse cell lines was compared by flow cytometry, using TMRM, Molecular Probes) as the probe (REF).
  • cells were harvested and resuspended at 1x10 cells/mL in cell culture medium containing 10 ⁇ M verapamil and 50 ⁇ M probenecid (SIGMA), MDR-1 and MRP-1 inhibitors respectively.
  • TMRM was added to each tube to a final 50 nM.
  • Cells were incubated at 37°C for 20 min. and immediately analyzed by flow cytometry.
  • NAO nonyl-acridine orange
  • A6 and control cells were seeded onto 25 mm-round glass coverslips at a density of 10 4 cells/coverslip and grown for 2 days prior to measurement. Cells were then stained with 20 nM TMRM dissolved in Hank's balanced salt solution (HBSS) with 10% FCS, 10 mM Hepes and 5 ⁇ M verapamil (Sigma) for 20 min at 37°C. Coverslips were placed on the stage of a Nikon Eclipse TE300 inverted microscope equipped with a Xenon-lamp illumination system and with a Hamamatsu Orca ER 12-bit digital cooled CCD camera.
  • HBSS Hank's balanced salt solution
  • FCS 10 mM Hepes
  • verapamil verapamil
  • Example 10 Mitochondrial stmcture and function are compromised in cells affected bv Fl 6 Electron-microscopy analysis was performed on untreated or F16-treated A6 cells, control cells and tumor cell lines SH1.1 and SMF to search for potential mitochondria lesions due to F16 accumulation. Cells grown in 35 mm dishes were left untreated or treated with 3 ⁇ M F16 for the indicated times, and fixed for 1 hr at room temperature in 0.1
  • mitochondria were the only organelles in which ultrastructural abnormalities were evident as early as 15 hr of F16 treatment.
  • A6 cells showed progressive swelling and disruption of mitochondrial integrity with concomitant mitochondrial membrane disruption, characteristic of apoptotic death.
  • no abnormalities in mitochondria mo ⁇ hology were observed.
  • ATP levels were determined in F16-treated cells by the luciferase-catalyzed ATP-dependent bioluminescent oxidation of luciferin using the
  • EpH4-EV and EpH4-A6 cells were grown in the absence or in the presence of 3 ⁇ M F16 for 15 hr and 24 hr. As a control A6 were treated with 5 ⁇ M FCCP (Sigma). Cells were then harvested, counted and equivalent numbers used for each determination, according to manufacturer's procedure. Each determination was done in triplicate, and the entire experiment was performed twice. The results, which are shown in Fig. 10, indicate that a significant depletion in the ATP pool was observed in cells after 24 hr of F16 incubation.
  • EpH4-EV and EpH4- A6 cells were grown in the absence or in the presence of 3 ⁇ M FI 6 for 15, 24, 48 and 72 hr. cells were resuspended and diluted to 10 6 cells/mL. DHE was added to the cell suspension to a final 2 ⁇ M in each case. Cells were incubated at 37°C for 15 min and immediately analysed using flow cytometry.
  • Mitochondria were isolated from liver of Balb/c7 mice by standard differential centrifugation, and resuspended in isolation buffer (0.2 M sucrose, 10 mM Tris-MOPS pH 7.4, 0.1 mM EGTA-Tris, 0.1% delipidated BSA) (Costantini et al. (1995) Toxicology (99) 77-88). Protein concentration was determined by Bradford assay.
  • Mitochondria (0.5 mg/ml) were incubated in experimental buffer with the indicated concentrations of F16 and changes in F16 fluorescence intensity were measured, using a LS-50B spectrofluorimeter at 25°C, with excitation and emission wavelengths set at 415 ⁇ 2.5 nm and 520 ⁇ 5 nm, respectively. All reactions were performed at 25°C and the instmment was equipped with magnetic stirring. The results indicate that F16 caused swelling of purified mitochondria in a dose- dependent fashion, and that cyclosporin A, a specific PTP inhibitor, completely abolished this effect. The mechanism of PTP induction by F16 was further investigated as follows.
  • PTP is a voltage dependent channel, with its open conformation being favored at lower membrane potential (Scorrano et al. (1997) Mol Cell Biochem (174) 181-4). It has previously been shown that DLCs such as chloromethyl tetramethyl rhodamine can cause mitochondrial depolarization (Scorrano et al. (1999) J Biol Chem (274) 24657-63). Therefore, it was investigated whether F16-mediated PTP opening could be the result of F16-induced mitochondrial depolarization. In principle, mitochondrial depolarization could be caused either by a direct protonophoric effect, i.e., by proton shuttling across the inner mitochondrial membrane, or indirectly as a consequence of inhibition of the respiratory chain.
  • F16 The fluorescent properties of F16 were used for this pu ⁇ ose. It was determined whether the fluorescence of a 1 ⁇ M solution of F16 changed in response to mitochondrial energization and depolarization. The results indicate that in response to mitochondrial energization, the fluorescence of the F16 solution increases, whereas membrane potential dissipation by FCCP completely reverts this fluorescence increase. Interestingly, when mitochondria were incubated with 50 ⁇ M F16, energization lead to a much smaller fluorescence increase. Preincubation of mitochondria with the PTP inhibitor CsA only partially blunted this effect. Thus, F16 directly causes mitochondrial depolarization and this depolarization is worsened by the secondary PTP opening. Taken together, these results show that F16 acts as a weak protonophore and that it causes PTP opening by lowering the mitochondrial membrane potential.
  • mitochondria As the molecular dissection of the apoptotic pathway proceeds, it becomes clearer that the cmcial role of mitochondria in propagating the cell death signal.
  • mitochondria In response to most proapoptotic stimuli, mitochondria release proteins- among them cytochrome c, apoptosis inducing factor, and repressors of caspase inhibitors such as Smac/DIABLO, from their intermembrane space (IMS) to the cytosol or the nucleus to amplify and execute the cell death program.
  • IMS intermembrane space
  • Fig. 12 The results indicate that the phosphotyrosine content of Neu, phosphorylated PKB/Akt, and phosphorylated MAP kinase are decreased in F16 treated cells.
  • Neu and PKB the protein level themselves were reduced as a consequence of F16 treatment. This downregulation was only partially blocked by the pan- caspase inhibitor Z-VAD-FMK.
  • previous studies have shown that Neu and PKB/Akt are substrates for caspase-mediated cleavage (Tikhomirov et al. J Biol Chem (276) 33675-80 and Bachelder et al. J Biol Chem (276) 34702-7).
  • Inhibition of proliferation was essentially specific to transformed cells, since the normal cells were inhibited only at concentrations of 10 ⁇ M or higher. This compound behaves similarly to FI 6, at least since it was also shown to accumulate in mitochondria of cancer cells and essentially not in mitochondria of normal cells.

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Abstract

L'invention concerne des composés et des procédés permettant de normaliser la prolifération cellulaire et/ou de moduler la différentiation cellulaire et/ou d'induire la mort cellulaire. Dans un mode de réalisation préféré, l'invention concerne des procédés permettant d'inhiber la prolifération de cellules hyperprolifératives, ces procédés comprenant l'opération consistant à mettre en contact les cellules avec une composition contenant une dose inhibitrice de croissance d'au moins un composé sélectionné dans le groupe comprenant E20 (ID 141525), F12 (ID 120590), F16 (ID 274873), H10 (ID 120670), J6 (ID 120856), N12 (ID 215015), L4 (ID 121113), B15 (ID 217496) et QR, ou des dérivés et analogues de ces composés, ou bien des sels pharmaceutiquement acceptables de ces composés.
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